In optical microscopy field the need of investigating samples, even of substantial thickness, is known, such as for example biological samples. Regions of interest are typically present inside the biological sample, such as for example specific cells. Such regions of interest can exhibit, above all in case of live biological samples, changes over time of their own optical features, corresponding to specific behaviors of the sample, whose detection is of interest for a biological analysis of the sample. In these cases, optical microscopy has to be able to detect both spatial information of regions of interests, namely where they are arranged inside the three-dimensional space under examination of the sample, and temporal information, namely the trend over time of optical features of the regions of interest.
In this technical field the document “Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging” (Sean Quirin, Darcy S. Peterka, and Rafael Yuste—Optics express 2013) discloses the sensing of spatial and temporal information about the sample by extended depth of field imaging. However spatial information is encoded on the basis of the particular configuration of the excitation path, that uses a two-photon system. Therefore, spatial information gets lost when using other excitation techniques. The document discloses the use of a micro-machined phase mask that provides a fixed phase modulation of the sensing path to have an extended depth of field. This means that the extended depth of field cannot be modified in amplitude, once the phase mask has been made and inserted. That is to say, this configuration does not allow the extension of the depth of field to be selected. The phase mask further induces aberrations in images that require deconvolution techniques to eliminate artifacts. Finally the extended depth-of-field acquisition method used does not include gathering spatial information of the sample in axial direction.
In the same technical field also the document “Rapid 3D light-sheet microscopy with a tunable lens” (Fahrbach et al. Optics express 2013) describes an analysis of spatial and temporal information of samples by optical microscopy. The document discloses a method using an electrically focus tunable liquid lens to perform a dynamic phase modulation in the reception path. Such lenses are currently known and available on the market and allow, by applying an electrical control signal, the curvature of the liquid to be controlled and therefore the focal length to be changed. This allows an arbitrary plane to be focused within a three-dimensional sample without moving any mechanical part of the system, and therefore allows a very rapid axial scanning of the detected in-focus plane to be performed without inertia. However, the document does not disclose the possibility of performing an extended depth of field imaging and therefore the method is limited in scan rate.
From the above it is clear that there is an unsatisfied need in prior art known methods for an optical microscopy method able to effectively obtain and analyse spatial and temporal information of samples, with high scan rates, with the possibility of setting the depth of investigation in the sample, and that does not require changes to the architecture of the used optical system.